"Chromatin cant be important otherwise bacteria wouldhave it." Comment made at a transcription meeting.

Transcriptioncontrol was once an understandable topic. The prevailingview was that transcription factors sought out and bound tospecific DNA sequences, thereby introducing activators or repressorsto particular target genes. Although these interactions coulderect elaborate castles on DNA, it was possible to considerthese edifices as a kind of simplistic "Lego model." For morethan a decade, transcription regulation was presented in cartoonrepresentations of ever-increasing Technicolor glory with DNAdrawn in a straight line. Slowly, the line began to bend asconcepts dealing with the structural packaging of DNA were considered.Today, an accumulated wealth of data has placed chromatin structurein a pre-eminent position in the field of transcription regulation.

It used to be that discussion of chromatin was relegated tothe postbanquet morning session at conferences. Attended byjust the die-hards and the remnants of the night before whohad not yet made it back to their rooms, the talks were oftenreplete with rigorous science, and they sparked intense discussion.From the field of epigenetic regulation, DNA methylation wasamong the first topics to emerge onto center stage and to befeatured in plenary talks, with histone acetylation close behind.The transcription factor "Lego" models adopted new components,and a loose coalition was formed between the transcription andchromatin fields. Now, it is quite respectable to discuss theimportance of chromatin structure, DNA methylation, and histonemodification to transcriptional control in many biological contextsand particularly in cancer. Therefore, it is worth some effortto consider the relative contributions and to examine the cooperativeinteractions among all components of the gene regulation machinery.This is particularly pertinent if we hope to intervene in themechanisms that control gene expression to correct the errorsthat result in oncogenic transformation. Here, we will reviewrecent progress toward understanding the role of DNA methylationin epigenetic regulation of gene expression, the interactionsbetween DNA methylation and other epigenetic systems that modulatechromatin structure, and the relevance of these topics to cancer.

In mammals, a major form of DNA modification involves methylationof the C5 position of cytosines within CpG dinucleotides (1, 2). Several studies have reported the presence of 5mC3
atnon-CpG sequences (2, 3, 4, 5, 6, 7, 8)
, and functional rolesfor these modifications have been proposed recently (3)
. However,for the purpose of this review, we will focus exclusively onthe processes and functional consequences of CpG methylation.

The distribution of CpG dinucleotides in mammalian genomes isnot random. Within coding regions, CpG occurs at a low frequency(1 CpG/100 bp), and these are predominantly methylated on bothstrands. However, the promoter and 5' transcribed sequencesof many genes include a region in which CpG occurs at or nearthe expected random frequency (1 CpG/10 bp). These "CpG islands"tend to be undermethylated, with the exceptions of CpG islandsthat are associated with transcriptionally silent alleles ofimprinted genes and silent genes on the inactive X chromosome(reviewed in Ref. 4
). Despite the higher frequency of CpG methylationat these loci, CpG islands associated with imprinted and X-inactivatedgenes account for <10% of total genomic 5mC. The bulk of5mC in the genome (70%) resides within CpG-rich transposonsscattered among extragenic and intronic sequences (5, 6)
.

The COOH-terminal region of Dnmt1 contains a series of motifscharacteristic of the known DNA (cytosine-5) methyltransferasesfrom bacteria to humans (7, 11, 12)
. These regions cooperateto form binding sites for the reaction substrates, S-adenosyl-L-methionine(AdoMet) and DNA, and the catalytic domain responsible for transferof the methyl donor group from AdoMet to C5 of the CpG dinucleotide.The precise mechanism of this enzymatic reaction has been reviewedelsewhere (10)
. Accumulating data have demonstrated that theNH2-terminal region of Dnmt1 comprises several important functionaldomains. These include a nuclear localization signal (13)
,a region that targets the protein to replication foci duringS phase (14, 15)
, sequences that partially reduce the de novomethyltransferase activity of the catalytic domain, and a cysteine-richzinc-binding domain (16, 17)
. As discussed below, the NH2-terminaldomain also includes sites of interaction with various proteinsinvolved in modulation of chromatin structure and gene regulation.

The enzymatic properties of Dnmt1 have been studied extensivelyusing in vitro biochemical assays as well as in vivo geneticapproaches. Although Dnmt1 can transfer a methyl group to symmetricallyunmethylated CpG dinucleotides in vitro, it preferentially methylateshemimethylated target sequences. The degree of this preferenceranges from 5- to 50-fold, depending on the specific study (9, 18,19, 20, 21, 22, 23)
, and little if any target sequencespecificity has been revealed outside the CpG dinucleotide itself(9)
. These findings suggest that Dnmt1 functions in the maintenanceof CpG methylation by methylating the daughter strand CpG afterreplication of symmetrically methylated loci.

Genetic studies in mice revealed that partial loss of Dnmt1function results in embryonic lethality. However, homozygousmutant ES cells are viable, and they exhibit no obvious growthor morphological abnormalities (24)
. Although these cells exhibitsubstantial demethylation of endogenous retroviral DNA, theyretain 30% of the normal level of genomic 5mC. Similar resultswere obtained with a complete loss-of-function dnmt1 allele(25)
. Although genomic 5mC content is reduced to levels significantlylower than those of ES cells expressing a partial loss-of-functiondnmt1 mutant, Dnmt1-null ES cells exhibit a low level of CpGmethylation. Furthermore, they retain the ability to de novomethylate newly integrated retroviral DNA. These studies confirmedthe existence of additional DNA (cytosine-5) methyltransferasesand, together with previous in vitro data, suggested that Dnmt1functions to maintain rather than to establish patterns of CpGmethylation.

Support for an exclusively maintenance function of Dnmt1 camefrom studies in which mammalian Dnmt was expressed in Drosophila(26)
, a species with only trace amounts of genomic cytosinemethylation (27, 28)
. Whereas expression of a mammalian denovo DNA methyltransferase (described below) increased cytosinemethylation, expression of exogenous Dnmt1 did not. Co-expressionof both enzymes resulted in a 31% increase in genomic 5mC contentrelative to flies expressing the de novo methyltransferase alone,possibly attributable to maintenance methyltransferase activityof Dnmt1 on hemimethylated substrates. These data indicate thatDnmt1 has no de novo activity in vivo. However, these studieswere carried out in cells that do not normally methylate DNAat a level comparable with that in mammalian cells. Thus, Drosophilacells may lack additional cofactors that direct proper de novoand maintenance cytosine methylation. A recent in vitro kineticstudy suggested that the zinc-binding domain of Dnmt1 preferentiallyinteracts with symmetrically methylated DNA, and addition ofsymmetrically methylated DNA stimulates zinc-dependent de novomethyltransferase activity (16)
. Thus, in mammalian cells,Dnmt1 may catalyze the spread of CpG methylation from regionswith pre-existing methylated CpG dinucleotides into nearby unmethylatedregions. This provides a plausible alternative explanation forthe lack of Dnmt1 de novo activity in Drosophila and the observationthat there is a cooperative effect of coexpression of Dnmt1and a de novo DNA methyltransferase. Regardless, the findingsoutlined above demonstrate that Dnmt1 represents the major mammalianenzyme responsible for maintenance of CpG methylation and thatit is complemented by one or more other enzymes capable of denovo methylation.

The identification of Dnmt3a and Dnmt3b confirmed the existenceof a family of mammalian DNA (cytosine-5) methyltransferases.These genes were identified in expressed sequence tag databasesby their sequence similarity with the catalytic domain of Dnmt1.However, the Dnmt3 proteins have no homology to Dnmt1 outsidethis region (21)
. The Dnmt3a locus encodes a single protein,whereas three alternatively spliced Dnmt3b isoforms have beendetected (21, 36)
. The Dnmt3 proteins contain a cysteine-richdomain related to the plant homeodomain present in many chromatin-associatedproteins. This region is most similar to the plant homeodomain-likedomain of ATRX (37)
, a member of the SNF2 family of helicase/ATPases(38, 39)
. Interestingly, mutations in ATRX cause X-linked-thalassemia mental retardation (ATRX) syndrome, which is associatedwith both hyper- and hypomethylation abnormalities of specificrepetitive elements (40)
.

The properties of the Dnmt3 family implicated these proteinsas the long-awaited de novo DNA (cytosine-5) methyltransferases.Unlike Dnmt1, neither Dnmt3a nor Dnmt3b shows a preference forhemimethylated DNA target sites in vitro(21, 36)
. Furthermore,the expression patterns of the Dnmt3 genes correlate with thetiming of developmental de novo methylation. Although Dnmt1is expressed ubiquitously in somatic cells, the Dnmt3 genesare expressed at a high level in undifferentiated embryonicstem cells but at low levels in differentiated somatic tissues(21)
. The de novo methyltransferase functions of Dnmt3a andDnmt3b have been confirmed by studies in genetically modifiedmice. Okano et al.(41)
produced embryonic stem cell lineswith homozygous null mutations in Dnmt3a and Dnmt3b, separatelyand in combination. Both single knock-out lines retained theability to methylate foreign retroviral DNA, whereas the doubleknock-out cells completely lacked this activity, demonstratingboth the requirement and the redundancy of Dnmt3a and Dnmt3bfor de novo methyltransferase activity. Despite their overlappingpatterns of expression and their largely redundant functions,the effects of independent loss of the two enzymes demonstratethat the two enzymes have distinguishable functions. Mice deficientin Dnmt3a survive to term, but they become runted and die at4 weeks of age. However, Dnmt3b-/- embryos develop normallybefore embryonic day (E) 9.5, but they die prior to term. Embryoslacking both enzymes show abnormal morphology by E8.5, and theydie by E11.5. Genomic methylation abnormalities in these embryosfurther demonstrate both overlapping and specific functionsof the Dnmt3 family. For example, C-type retroviral DNA andintracisternal A particle repeats are unaffected (Dnmt3a-/-)or only slightly undermethylated (Dnmt3b-/-) in single knock-outembryos, but they are substantially undermethylated in doubleknock-out embryos. However, the overall methylation level indouble knock-out embryos remains higher than the level in embryoslacking Dnmt1. Centromeric minor satellite repeats are significantlydemethylated in Dnmt3b-/- cells but unaffected in Dnmt3a-/-cells, suggesting that this class of sequence is methylatedspecifically by Dnmt3b.

The identification of mutations responsible for ICF (immunodeficiency,centromeric region instability, and facial anomalies) syndromeprovided a natural demonstration of target specificity for Dnmt3b(41, 42, 43)
. This genetic disorder is characterized cytogeneticallyby marked hypomethylation of specific classical satellite repeats(44, 45)
, elongation of juxtacentromeric heterochromatin inlymphocytes, and various structural abnormalities involvingchromosomes 1, 9, and 16 (46)
. Therefore, although none ofthe known DNA (cytosine-5) methyltransferases show target specificityin vitro, mechanisms exist to direct their activity to appropriategenomic loci in vivo. This is currently an exciting area ofresearch, and as discussed below, the recent identificationof key molecules is beginning to shed light on critical pathwaysthat regulate DNA methylation.

The correlation between DNA methylation and transcriptionalinactivity is well established. However, a causative role forCpG methylation in repression of transcription has often beena subject for debate. Although many silenced genes are associatedwith dense CpG methylation, this epigenetic mark could be adownstream consequence of transcriptional inactivity ratherthan an active participant in the process of repression. However,the identification of a family of proteins that bind to DNAcontaining methylated CpG dinucleotides established a causativelink between CpG methylation and repression of transcription.The members of this family have been comprehensively reviewed(47, 48, 49, 50)
. Therefore, we will briefly discuss the generalcharacteristics of the mammalian family members.

The presence of methyl-CpG-binding proteins in human cell extractswas demonstrated nearly two decades ago (51)
. MeCP2 was thefirst individual component of these complexes to be purifiedand biochemically characterized (52, 53)
. This protein containsan NH2-terminal MBD (54)
and a COOH-terminal TRD (55)
. Itassociates with chromatin (53)
and localizes to methyl-CpG-richsequences in vivo(56)
. In vitro, the intact protein or theMBD alone selectively binds to DNA containing symmetricallymethylated CpG dinucleotides with an affinity directly proportionalto methyl-CpG density (52, 57)
. Likewise, the TRD repressestranscription independently of other MeCP2 sequences (54, 58,59, 60)
. Binding of the Sin3a corepressor to the TRD recruitsHDAC into the complex (58, 59)
. These findings provide a conceptualframework for a chain of events by which DNA methylation activelypromotes transcriptional silencing. According to the model,MeCP2 binds to chromosomal regions containing methylated CpGdinucleotides. A histone deacetylase corepressor complex isthen recruited by the binding of Sin3a/HDAC to MeCP2. Histonedeacetylation, in turn, results in condensation of chromatinleading to a local chromatin structure that is refractory toinitiation of transcription (reviewed in Ref. 50
). Consistentwith this model, in vitro transcription repression by MeCP2is sensitive to histone deacetylase inhibitors (58, 59)
. However,the complexity of interactions among members of both the methyltransferaseand MBD families suggests that this pathway may be one of severalintersecting pathways leading to methylation-dependent transcriptionrepression. In fact, there is evidence for HDAC-independentmechanisms of transcription repression by MBD proteins, furtherextending the potential impact of DNA methylation on gene expression(47, 60, 61, 62)
. Furthermore, in colorectal carcinoma andin leukemic cell lines, hypermethylated, transcriptionally silentgenes can be reactivated by simultaneous treatment with theHDAC inhibitor TSA and the demethylating agent 5-Aza-dC butnot by TSA alone (63)
. These findings suggest either that DNAmethylation has additional repressive effects that are independentof histone deacetylation, or that unknown TSA-insensitive HDACsare also involved.

Additional MBD-containing proteins have been identified throughan in silico approach to cloning (64, 65)
. Five MBD familymembers have now been identified. MBD1 has an NH2-terminal MBDand a COOH-terminal TRD. In addition, full-length MBD1 containsthree CXXC motifs similar to the motif present in Dnmt1 (64)
.MBD1 binds preferentially to densely methylated DNA in vitro,and it represses transcription in a HDAC-dependent manner intransfected cells (64, 66, 67)
. Consistent with its in vitroDNA binding characteristics, overexpressed green fluorescentprotein-tagged MBD1 localizes to densely methylated major satelliteDNA in mouse cells (65)
, and it is concentrated at methylatedpericentromeric regions of chromosome 1 in human cells (68)
.Endogenous MBD1 is detected along euchromatic regions in humandiploid metaphase chromosome spreads, but it concentrates atcentromeric heterochromatic regions of chromosomes 1, 9, 15,and 16, as well as regions of densely methylated spacer DNAsequences interspersed among rRNA genes. Furthermore, the intensityof MBD1 staining is generally inversely proportional to thestaining intensity of acetylated histone H4 (66)
. In humancells, MBD1 mRNA is expressed as five alternatively splicedforms that encode isoforms differing in their COOH-terminaland CXXC regions (65, 68)
. Although the functional consequencesof these alternative forms are unclear, inclusion of all threeCXXC motifs results in an MBD1 isoform capable of repressingtranscription independently of DNA methylation in transfectedcells (67, 68)
.

MBD2 includes partially overlapping MBD and TRD domains (69)
.The MBD binds to DNA with a single methylated CpG in vitro(65, 70), and an MBD2-GFP fusion protein binds to major satelliteDNA in transfected mouse cells (65)
. Consistent with an HDAC-dependentmodel of gene repression, the TRD exhibits TSA-sensitive transcriptionrepression activity in reporter assays (62)
. Ng et al.(62)identified MBD2 as the methyl-binding component of the MeCP1complex, a methyl-CpG-binding activity that is distinguishablefrom MeCP2 in that MeCP1 requires more densely methylated DNAfor binding (71)
. MeCP1 was recently suggested to comprisea chromatin remodeling ATPase (NuRD) complex with associatedHDAC activity (62, 72)
. Furthermore, MBD2 has been reportedto be associated with Sin3a (69)
. It is likely that the variationin reported factors and complexes associated with MBD2 reflectsseveral distinct yet overlapping cell context-dependent functionsof this family of proteins.

MBD3 has extensive sequence similarity to MBD2 (65)
. It isexpressed as several splice variants, some of which disruptthe MBD (65, 73)
. The protein has been identified as a componentof the Mi-2/NuRD transcriptional corepressor complex that includesMi-2 ATPase, HDAC, and other proteins (70, 73, 74)
. However,in vitro, mammalian MBD3 has little if any methyl-CpG-bindingactivity, likely because of amino acid substitutions withinthe MBD (65, 70)
. Therefore, unlike the case for the Xenopusorthologue of MBD3 which contains a MBD with methyl-CpG-bindingactivity (70)
, it is unlikely that mammalian MBD3 plays a rolein methylation-dependent transcription repression.

Finally, MBD4 includes a MBD similar to that of MeCP2, althoughthe COOH-terminal domain is homologous to bacterial DNA repairenzymes (65)
. Although MBD4 is capable of binding to methyl-CpGsites, it has a higher affinity for 5mCpG-TpG mismatched sites(75)
, and the DNA repair domain provides DNA N-glycosylaseactivity at G-T mismatches (75, 76)
. Therefore, MBD4 is ideallysuited to function in the repair of point mutations that resultfrom spontaneous deamination of 5-methylcytosine to thymine.In addition, MBD4 (also known as MED1) binds to the MLH1 DNAmismatch repair protein in vivo. Expression of a MBD4 mutantlacking the MBD induces microsatellite instability in cell lines,implicating MBD4 in this form of DNA repair as well (77)
. Thesedata suggest that MBD4 may serve as a strand discriminationfactor for MLH1, directing mismatch repair activity to the newlysynthesized strand. However, in an in vitro assay, nuclear extractscontaining MBD4 perform mismatch repair independently of targetCpG methylation status (78)
.

Collectively, the studies reviewed above have contributed toa basic understanding of the enzymes that establish and maintainCpG methylation, as well as mechanisms by which these epigeneticsignals are interpreted. However, much remains to be sortedout, and recent findings have added additional layers of complexityto the pathways involved. A productive approach toward understandingthe regulation of DNA methylation has been the search for proteinsthat interact with the methyltransferases (reviewed in Ref.79
). Because Dnmt1 was the first mammalian (cytosine-5) methyltransferaseto be identified, it remains the most extensively studied todate. PCNA, a DNA polymerase processivity factor required forDNA replication (80)
, binds to an NH2-terminal region of Dnmt1(15)
. PCNA is recruited to sites of replicating DNA by CAF-1p150, a factor responsible for assembly of nucleosomes ontoreplicating DNA (81)
. The interaction of PCNA with Dnmt1 thereforeprovides an attractive mechanism by which Dnmt1 is directedto sites of newly replicating DNA to maintain full methylationstatus after replication (82)
.

In addition to the indirect recruitment of HDACs via MBD proteins,Dnmt1 interacts directly with HDAC1 and HDAC2 (83, 84, 85)
.This interaction may provide an additional association betweenDNA methylation and chromatin condensation by bringing the factorsrequired for both into proximity. The association of HDAC withMBD proteins may then serve a maintenance-repressive role bykeeping histones in a deacetylated state independently of Dnmt1.As discussed below, factors involved in histone modificationand chromatin remodeling may also establish a local chromatinstructure that provides the DNA methylation machinery accessto DNA targets.

Rountree et al.(85)
identified a novel protein, DMAP1, thatbinds to the extreme NH2-terminus of Dnmt1. When fused to ageneric DNA binding domain, DMAP1 functions as an HDAC-independenttranscriptional repressor, possibly accounting for at leastsome of the HDAC-independent repression ability of Dnmt1 (83, 85). Dnmt1 has also been shown to exist in complex with thepRb tumor suppressor and the pRb-associated E2F-1 transcriptionalactivator in vivo(84)
. In nondividing cells, pRb binds toE2F-1 and represses transactivation of genes involved in cellcycle progression (86)
. Additionally, pRb binds to HDAC (87)
.The interaction of Dnmt1 with pRb may enhance its repressiveeffects by targeted methylation of E2F-1 binding sites or byrecruitment of histone deacetylases to these loci.

These discoveries demonstrate that Dnmt1 is linked to severalpathways associated with transcription repression. Ongoing studiesof the other DNA (cytosine-5) methyltransferase family memberswill certainly provide additional insights into the complexrelationship between these enzymes and the regulation of geneexpression. In fact, Fuks et al.(88)
recently demonstratedthat Dnmt3a binds RP58, a transcriptional repressor associatedwith heterochromatin and with promoters of various tissue-specificgenes, potentially providing a mechanism for sequence-specifictargeting of the enzyme (89)
. Dnmt3a also directly interactswith HDAC1 (88)
.

Although the effect of CpG methylation on transcription is mediatedlargely by histone-modifying factors, it is now apparent thatchromatin remodeling factors are also involved in regulationof the global methylation pattern. In Arabidopsis thaliana,the ddm1 (decrease in DNA methylation 1) mutant exhibits a 70%reduction of genomic cytosine methylation involving mostly repetitivesequences (90)
. Decreased methylation of low-copy sequencesoccurs over multiple generations (91)
. DDM1 is a member ofthe SNF2-like ATPase/helicase family of proteins that catalyzeATP-dependent disruption of histone-DNA interactions (92)
.A model was proposed in which nucleosome remodeling is essentialfor the establishment and maintenance of DNA methylation, possiblyby allowing the methylation machinery access to DNA targets(Fig. 1)
. An analogous system was subsequently identified inmammals. Homozygous disruption of lymphoid-specific helicase,Lsh (also known as PASG, proliferation-associated SNF2-likegene), results in perinatal lethality in mice (93)
and 50%reduction in global cytosine methylation content affecting repetitiveelements and CpG islands associated with select imprinted alleles(94)
. Lsh, which is ubiquitously expressed primarily duringS-phase, is closely related to the SNF2 subfamily. It containsATPase and helicase domains similar to those of ArabidopsisDDM1 and other proteins in yeast, mice, and humans (reviewedin Ref. 95
).

Recent studies of DNA methylation in Neurospora crassa may alsoprovide insight into the regulation and functional role of DNAmethylation in mammals. In a genetic screen for mutants withdecreased genomic cytosine methylation, Kouzminova and Selker(96)
identified dim-2, a DNA methyltransferase responsiblefor both de novo and maintenance cytosine methylation. A subsequentinsertional mutagenesis strategy fortuitously generated a mutationin an independent gene, dim-5, that is also essential for cytosinemethylation. Surprisingly, this gene encodes a protein methyltransferasethat specifically methylates lysine 9 of histone H3 (H3-Lys9;Ref. 97
). The protein is homologous to the H3-Lys9 methyltransferasesof Saccharomyces pombe (Clr4), Drosophila, and humans (Suv39h;Ref. 98
). Consistent with the requirement of H3-Lys9 methylationfor DNA cytosine methylation, expression of histone H3 mutantswith amino acid substitutions of Lys9 results in decreased genomiccytosine methylation in a wild-type strain (97)
. Adding yetanother level of regulation to the system, methylation of histoneH3-Lys9 is itself an epigenetic mark of heterochromatin. Themechanism involves binding of heterochromatin protein 1 (HP1,reviewed in Refs. 99
, 100
) to Lys9-methylated histone H3,resulting in chromatin remodeling and gene silencing (101, 102,103, 104)
. If there is also a requirement for histone H3-Lys9methylation for DNA methylation in mammalian cells, this suggeststhat an interplay of DNA and protein modifications may mediatethe establishment and maintenance of DNA methylation as wellas the epigenetic modulation of transcription repression (Fig.1)
. The ultimate target of these events is the nucleosome.For example, ATP-dependent nucleosome remodeling factors (i.e.,Lsh) may be required to loosen DNA-histone contacts, facilitatingaccess of DNA (cytosine-5) methyltransferases to target DNAsequences. Simultaneously, histone methyltransferases (i.e.,Suv93H1) may promote establishment of nucleoprotein complexesthat recruit DNA (cytosine-5) methyltransferases to target locior remodel chromatin into conformations permissive for DNA methylation.Alternatively, these processes may be linked to protection againstDNA demethylation rather than the establishment or maintenanceof DNA methylation. Finally, the epigenetic territorial markers,CpG and H3-Lys9 methylation, also recruit factors that furthermodify histones, resulting in locally condensed, transcriptionallysilent heterochromatin.

The studies summarized above suggest that disruptions withinchromatin modification pathways affect highly repetitive, constitutiveheterochromatin. However, precise epigenetic patterns must alsobe established at low-copy loci. For example, CpG methylationand histone H3-Lys9 methylation associate with the facultativeheterochromatin of the inactive X chromosome and promoters ofspecific X-inactivated genes (105, 106)
. Furthermore, reminiscentof its potential targeting function for Dnmt1, pRb has recentlybeen shown to bind to both Suv39H1 and HP1, directing theiractivities to the cyclin E promoter (107)
. Finally, geneticinactivation of an unconventional member of the Dnmt familyrevealed a role in targeting de novo methylation. Dnmt3L hasextensive homology to Dnmt3a and Dnmt3b, but it lacks a functionalDNA methyltransferase catalytic domain (108)
. Bourchiset al.(109)
recently produced mice lacking functional Dnmt3L.This mutation behaves as a maternal effect lethal. Homozygousmutant males are viable, but sterile. Homozygous females arefertile, yet their heterozygous offspring die before term. Surprisingly,these mutant embryos exhibit a very specific DNA methylationabnormality. They lack the ability to properly methylate maternallyrepressed imprinted genes (Snrpn and Peg1), yet they maintainproper allele-specific methylation of the paternally methylatedH19 gene. Therefore, Dnmt3L is required for the de novo establishment,but not maintenance, of specific genomic imprints. It is likelythat Dnmt3L functions not as a methyltransferase but insteadas a regulatory cofactor that directs the activity of othermethyltransferase(s) to appropriate targets.

These findings raise many additional questions concerning theinterdependence of pathways leading to chromatin remodelingand regulation of transcription. Such elaborate regulatory networkspresent many potential points of deregulation (Fig. 2)
. Furtherelucidation of the mechanisms involved will likely increaseour understanding of the process and consequences of CpG methylation,and they may uncover currently unrecognized avenues leadingto tumorigenesis.

At a simplistic level, tumorigenesis arises as a consequenceof two related events, increased activity of factors that promotecell proliferation and decreased activity of factors that suppressunchecked proliferation. These basic scenarios demonstrate theinvolvement of both activating and inactivating mechanisms intumorigenesis. However, additional abnormalities must accompany(or even supercede) changes in proliferative capacity for atransformed cell to cause malignancy. For example, transformedcells acquire the ability to invade surrounding tissue and travellong distances to establish themselves in new environments.They remodel local vasculature to feed aggressively growingcells, they circumvent intrinsic surveillance cell death mechanisms,and they impair DNA repair systems. Therefore, cancer involvesnot only aberrant proliferation but also subversion of mechanismsinvolved in the regulation of cell-cell and cell-matrix attachment,growth factor signaling, apoptosis, and recombination. In termsof inactivation of tumor suppressor genes, Knudsons "two-hit"hypothesis is a cornerstone concept. DNA mutations and chromosomalloss or rearrangements have traditionally received the mostattention. However, given the number of pathways altered duringcreation of an environment permissive for tumorigenesis, processeslinked to DNA methylation, provide additional potential mechanismsleading to heritable genomic changes that promote cancer.

The discovery of global CpG methylation abnormalities in tumorsled to the initial implication of a role of DNA methylationin cancer (110, 111)
. Tumor cells may harbor simultaneoushypermethylation of specific CpG islands and global hypomethylationof widespread transposon elements. Potentially, both eventsplay active roles in tumorigenesis. Although precise mechanismshave not yet been demonstrated, DNA methylation may protectthe genome by inhibiting homologous recombination between highlyrepetitive sequences (112)
. Therefore, loss of DNA methylationat these loci may increase the frequency of inappropriate recombinationleading to chromosomal abnormalities prevalent in tumors. Thishypothesis is supported by the finding that ES cells lackingfunctional Dnmt1 have a 10-fold higher mutation rate involvinggene rearrangements than wild-type ES cells (34)
. Because cytosinemethylation increases the frequency of C-to-T point mutationsattributable to deamination of 5mC to uracil, CpG methylationmay play a passive role in promoting point mutations. For example,a high frequency of p53 mutations occur at presumably methylatedexonic CpG sites (113, 114)
. Regarding hypermethylation ofspecific CpG islands, several transcriptionally silent genesexhibit dense CpG island methylation in tumors and tumor celllines, suggesting that these events either initiate transcriptionsilencing, or they participate in maintaining genes in a repressedstate. As reviewed by Baylin and Herman (115)
, nearly halfof the tumor suppressor genes carrying germ-line mutations infamilial cancers have been shown to be inactivated in associationwith CpG island hypermethylation in sporadic cancers. Theseinclude VHL, p16INK4a, pRb, ARF/INK4a, and several others. Equallytelling, the list to date includes genes whose protein productsparticipate in many processes required to create a microenvironmentsuitable for tumorigenesis and metastasis. Among these are genesthat function in suppression of invasion (E-cadherin, mts-1,and others), inhibition of angiogenesis (Thrombospondin-1, TIMP3),apoptosis (DAPK1, Fas) and DNA protection or repair (O6-MGMT,hMLH1, GSTP1, and BRCA-1). Genes silenced in association withhypermethylation in various tumor types have been reviewed (79, 115,116, 117)
. This group likely represents only a fractionof the aberrant methylation events potentially important incancer. Furthermore, the current list probably reflects an ascertainmentbias toward genes with previously demonstrated roles in tumorigenesis,because of the candidate gene approach traditionally used foridentification of methylation abnormalities. Consequently, unbiasedapproaches for identification of methylation changes associatedwith cancer have been established recently.

Within the past few years, microarray-based approaches haveallowed simultaneous analysis of mRNA expression levels representingthousands of genes. Likewise, array-based strategies are beingadapted for high-throughput analysis of DNA methylation patterns(129, 130)
. Although these technologies are in their infancy,the implications are evident. One can envision a future in whichglobal signatures of the epigenome, transcriptome, and proteomeare mapped for various conditions, providing unprecedented toolsfor cancer diagnostics, prognostics, and therapeutics.

The initial proposal that DNA methylation plays a direct rolein transcriptional regulation was met with skepticism. Similarly,the concept that aberrant DNA methylation plays a direct rolein tumorigenesis was not immediately embraced by the field.It is now clear that DNA methylation can actively participatein transcriptional repression in several ways. Furthermore,hypermethylation of CpG islands associated with transcriptionallysilent genes is featured in many tumors. However, questionsremain regarding the causative role of CpG island hypermethylationin tumorigenesis. Because tumor cells represent the final stateof a complex process leading to cancer, these questions areoften difficult to answer. Several observations suggest thatDNA methylation abnormalities represent more than simple markersof transformation. For example, in particular familial or sporadiccancers, hypermethylation is the sole detectable explanationfor complete loss of expression and activity of pRb, VHL, BRCA1,or p16INK4a tumor suppressor genes (reviewed in Refs. 79
, 115
,
)). Using a high-throughput candidate gene approach based onmethylation-specific PCR, Esteller et al.(131)
found thattumors from patients with inherited cancers in which one alleleof a particular tumor suppressor gene was mutated and the remainingallele was lost, the retained mutant allele was never hypermethylated.However, in tumors in which both the mutated and the wild-typeallele were retained, DNA hypermethylation served as a frequent"second hit" to inactivate the functional allele. These findingsdemonstrate a powerful selective force for methylation-associatedinactivation independent of genetic mutation (131)
. Other examplessuggest that DNA hypermethylation can actually promote geneticinstability. The DNA repair gene hMLH1 is frequently hypermethylatedin sporadic colorectal carcinomas with microsatellite instability.Treatment of cell lines derived from these tumors with the demethylatingagent 5-Aza-dC results in re-expression of hMLH1 and partialrestoration of mismatch repair activity (132)
. Finally, CpGmethylation changes can occur very early in tumorigenesis. Hypermethylationof p16INK4a in lung cancer has been detected in preneoplasticcells, the degree of which is directly proportional to diseaseprogression (133, 134, 135)
.

Although these findings establish a strong correlation betweentumorigenesis and hypermethylation of specific tumor suppressorgenes, confirmation of a causative relationship awaits demonstrationof the precise mechanisms of aberrant DNA methylation and analysisof their functional consequences throughout the process of cellulartransformation. One potential mechanism is up-regulated expressionof the DNA (cytosine-5) methyltransferase themselves. Kautiainenand Jones (136)
demonstrated increased DNA methyltransferaseactivity in various tumorigenic cell lines relative to nontumorigeniccells. Subsequently, increased DNA methyltransferase activitywas shown to coincide with increased expression of DNMT1 inhuman neoplastic cells and tumor tissues, and the magnitudeof expression increased with progressive stages of disease (137, 138). Forced overexpression of a mouse Dnmt1 cDNA results inincreased genomic DNA methylation and induction of cellulartransformation and tumorigenic potential of NIH 3T3 cells (139)
.In fibroblasts transformed by overexpression of human DNMT1,de novo methylation of particular CpG islands occurs within70 population doublings (140)
. More recently, elevated expressionof the DNMT3b (141)
and decreased expression of MeCP2 and MBD2(142)
in tumors have been reported. Furthermore, in some colorectalcarcinomas with microsatellite instability, frameshift mutationsarising from small insertions or deletions within an (A)10 tractin the coding region of MBD4 have been detected (143, 144)
.The frameshift mutations could lead to expression of a truncatedform of MBD4 that includes the MBD but lacks the DNA repairdomain. This could potentially enhance mutagenesis in tumorswith microsatellite instability because of a dominant-negativeeffect of the truncated protein (143)
or complete inactivationof MBD4 function by loss of heterozygosity (144)
.

Evidence for a functional role of Dnmt1 in tumorigenesis hasalso been obtained using genetically modified mice. The Minmouse, a valuable model of intestinal cancer, is heterozygousfor a germ-line mutation in the adenomatous polyposis coli (Apc)gene. Human APC is mutated (145)
or associated with promoterhypermethylation (146)
in the majority of colon cancers, adisease in which promoter hypermethylation of several geneshas been reported. On the B6 mouse genetic background, heterozygosityfor the Min allele predisposes to development of hundreds ofintestinal adenomas (147)
. Laird et al.(148)
demonstratedthat crossing B6-Min/+ mice to mice with one mutated Dnmt1 allele(129/SvJ-Dnmt1S/+) results in a dramatic decrease in the frequencyof intestinal adenomas. Early treatment of these mice with 5-Aza-dCresulted in further reduction of tumor incidence by nearly 60-fold(148)
. By crossing B6-Min/+ mice to the homogeneous B6-Dnmt1N/+strain, Cormier and Dove (149)
found that the effect of Dnmt1deficiency on tumor incidence and growth is independent of thestatus of p53 or modifier of Min 1 (Mom1), two loci that conferstrong resistance to Min-induced intestinal tumorigenesis. Interestingly,although Dnmt1 deficiency and Mom1 affect tumorigenesis independently,together they act synergistically to reduce tumor incidenceby >40-fold, and they completely prevent tumor developmentin nearly half of the mice studied (149)
.

Mechanistically, overexpression of Dnmt1 has been associatedwith transforming oncogenes including ras(150, 151)
, SV40large T-antigen (152)
, and fos(153)
, suggesting that increasedexpression of Dnmt1 may play a role in cellular transformation.In these model systems, up-regulation of Dnmt1 expression andactivity appears to be necessary for complete cellular transformationbecause antisense inhibition of Dnmt1 expression leads to restorationof nontransformed cellular morphology and growth properties(152, 153, 154, 155)
, and it decreases tumorigenic growth oftransformed cells in syngeneic mice (154, 155)
. Additionally,treatment of fos-transformed fibroblasts with TSA results inreversion to a more normal cellular morphology, implicatingchromatin remodeling in aspects of the transformed phenotype(153)
. Importantly, steady-state Dnmt1 mRNA levels vary duringthe cell cycle, increasing in parallel with proliferation (156)
.Therefore, elevation of Dnmt1 levels has been proposed to simplyreflect the increased proliferative capacity of transformedcells (10, 157, 158)
. However, studies of the role of Dnmt1in cellular transformation by fos demonstrated that increasedexpression and activity of Dnmt1 can be uncoupled from cellcycle regulation. Enforced expression of fos in cultured fibroblastsresults in morphological cellular transformation independentlyof cell proliferation (159)
. In growth-arrested fibroblasts,ectopic induction of c-fos expression results in morphologicaltransformation, increased expression of the endogenous Dnmt1gene, elevated DNA methyltransferase activity, and increasedgenomic 5mC content (153)
. When ectopic expression of c-fosis repressed, these cells revert to a nontransformed morphology,Dnmt1 expression and activity decrease, and genomic 5mC contentreturns to basal levels. Intriguingly, these results demonstratethat genomic 5mC content can be both increased and decreasedindependently of cell division, implying that an active demethylationprocess functions during reversion of fos transformation. Indeed,an isoform of the MBD2 protein has been reported to possess5mC demethylating activity in vitro(160)
. This finding impliesthat MBD2 may possess bimodal functions involving both methylation-dependenttranscriptional repression and modulation of precise DNA methylationpatterns. However, the demethylating ability of MBD2 has beendisputed (62, 70)
.

These results demonstrate a potential proliferation-independentrole for increased Dnmt1 activity during oncogenic transformation.However, because transformation requires continued oncogeneexpression, the relevant defect may lie in sustained activityof DNA (cytosine-5) methyltransferases within an inappropriatecellular context rather than in the absolute level of Dnmt1.Consistent with this possibility, DNA methyltransferase activitydecreases during G0-G1 arrest of normal bladder fibroblasts,yet bladder tumor cells maintain a higher level of methylationactivity throughout the period of growth arrest (161)
. Furthermore,the Dnmt1 isoform expressed in tumors has not been shown toindependently induce cellular transformation or tumorigenesis.The studies described above in which ectopic expression of Dnmt1induced cellular transformation and tumorigenesis used an NH2-terminallytruncated form of Dnmt1 (Dnmt1o) corresponding to the oocyte-specificversion of the enzyme (139, 140, 153)
. In our hands, overexpressionof the full-length somatic isoform of Dnmt1 (Dnmt1s) inducescell death rather than cellular transformation.4
Yet, duringcellular transformation by ras, SV40 large T-antigen or fos,the endogenous somatic Dnmt1 isoform is necessary for full cellulartransformation (152, 153, 154, 155)
and tumorigenic potential(154, 155)
. Therefore, it appears that deregulation of Dnmt1activity participates in transformation only in cells in whichoncogenic pathways have been activated. This implies that themechanisms involving Dnmt1 (including but not restricted toDNA methylation) must interact with additional cellular processesto participate in transformation. The fact that the oocyte-specificform of Dnmt1, which lacks an NH2-terminal region that participatesin various protein-protein interactions, can induce transformationand tumorigenesis may direct attention toward converging pathwayslinking Dnmt1 to mechanisms of tumorigenesis.

A recent study of the mechanisms of transcription repressionby PML-RAR suggests a link between DNA (cytosine-5) methyltransferasesand an oncogenic transcription factor fusion that is independentof the level of Dnmt1 expression (162)
. In >90% of APL cases,a reciprocal chromosomal translocation involving PML and theRA receptor RAR leads to expression of an oncogenic PML-RARfusion transcription factor (163, 164)
. PML-RAR functionsas a repressor of RA target gene transcription through a mechanisminvolving recruitment of an HDAC complex, resulting in a blockin the differentiation of APL blasts (165)
. Di Croce et al.(162)
found that induction of PML-RAR results in increasedCpG methylation in the 5' region of the RA receptor RARß2,a gene repressed by PML-RAR. Physical interactions between PML-RARand either Dnmt1 or Dnmt3a were detected by coimmunoprecipitationin an inducible PML-RAR expression cell line and in APL-derivedcells. In the presence of PML-RAR, Dnmt1 and Dnmt3a are recruitedto the RARß2 promoter, and the proteins colocalizein transfected cells. Furthermore, RA and 5-Aza-dC act synergisticallyto reduce RARß2 CpG methylation and reactivate RARß2expression in APL-derived cells (162)
. These results suggestthat repression of gene expression by oncogenic PML-RAR involvesrecruitment of Dnmts to target loci as well as direct associationwith HDAC complexes. The additional interactions among Dnmts,MBDs, and HDACs assembled at these loci may cooperate to ensuretranscriptional repression of putative tumor suppressor genes.

Although the relationships between DNA methylation, chromatinremodeling, and transcription repression have now been establishedconvincingly, conflicting observations still fuel skepticismregarding the role of DNA methylation in cancer. Overexpressionof DNA (cytosine-5) methyltransferases is not invariably detectedin tumor tissues in which DNA methylation abnormalities exist.Tumor cells exhibit both DNA hyper- and hypomethylation abnormalities,making mechanisms based on increased methyltransferase activitydifficult to reconcile. Furthermore, somatic inactivation ofDNMT1 by homologous recombination in human colorectal carcinomacells leads to a dramatic decrease in DNA (cytosine-5) methyltransferaseactivity; yet, these cells maintain the hypermethylated statusof the p16INK4a tumor suppressor gene, demonstrating that Dnmt1is not necessary for maintenance of this DNA methylation abnormality(166)
. Regarding these points, it is important to keep in mindthat the properties of tumor cells represent an end-state ofa process, and they do not necessarily reflect the combinationof abnormalities that participate in tumor development. Therefore,DNA (cytosine-5) methyltransferase levels and global methylationpatterns in tumor cells do not necessarily reflect their progressiveabnormalities that contributed to the evolution of the tumor.As reviewed above, CpG methylation patterns are influenced bynumerous cooperating pathways, including de novo and maintenancemethylation, demethylation, and factors that may direct or preventmethylation of appropriate and inappropriate targets, respectively.Cancer is a disease involving both genetic and epigenetic abnormalities.Numerous cellular events promote mutagenic "hits" within genetargets that initiate or promote tumorigenesis. Likewise, abnormalitieswithin the complex pathways that are linked to DNA methylationcan lead to several simultaneous "hits" within the epigenome,altering expression of numerous genes by modulating chromatinstructure (Fig. 2)
. The current explosion of data relatingto the complex pathways that target, maintain, and interpretepigenetic information encoded by DNA methylation promises amore comprehensive understanding of the process of tumorigenesis.Bacteria may not have chromatin, but they do not get cancereither.

Acknowledgments

We thank David Benhayon, Hiromichi Kimura, and Stysia Romerfor critical review of the manuscript.

Footnotes

The costs of publication of this article were defrayed in partby the payment of page charges. This article must thereforebe hereby marked advertisement in accordance with 18 U.S.C.Section 1734 solely to indicate this fact.

Fatemi M., Hermann A., Pradhan S., Jeltsch A. The activity of the murine DNA methyltransferase Dnmt1 is controlled by interaction of the catalytic domain with the N-terminal part of the enzyme leading to an allosteric activation of the enzyme after binding to methylated DNA. J. Mol. Biol., 309: 1189-1199, 2001.[Medline]